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Nrf2 protects against long-term PM2.5 exposure-induced liver inflammation by
positively regulates SIKE activation: Intervention by Juglanin
Chenxu Ge, Jun Tan, Shaoyu Zhong, Lili Lai, Geng Chen, Junjie Zhao, Chao Yi,
Longyan Wang, Liwei Zhou, Tingting Tang, Qiufeng Yang, Deshuai Lou, Qiang Li,
Yekuan Wu, Linfeng Hu, Gang Kuang, Xi Liu, Bochu Wang, Minxuan Xu
PII: S2213-2317(20)30850-8
DOI: https://doi.org/10.1016/j.redox.2020.101645
Reference: REDOX 101645
To appear in: Redox Biology
Received Date: 31 May 2020
Revised Date: 6 July 2020
Accepted Date: 11 July 2020
Please cite this article as: C. Ge, J. Tan, S. Zhong, L. Lai, G. Chen, J. Zhao, C. Yi, L. Wang, L. Zhou,
T. Tang, Q. Yang, D. Lou, Q. Li, Y. Wu, L. Hu, G. Kuang, X. Liu, B. Wang, M. Xu, Nrf2 protects
against long-term PM2.5 exposure-induced liver inflammation by positively regulates SIKE activation:
Intervention by Juglanin, Redox Biology, https://doi.org/10.1016/j.redox.2020.101645.
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Graphical abstract
Nrf2 protects against long-term PM2.5 exposure-induced liver inflammation by
positively regulates SIKE activation : Intervention by Juglanin
Chenxu Ge
a, b, c
*, Jun Tan
a, c
*
#
, Shaoyu Zhong
a
, Lili Lai
a
, Geng Chen
a
, Junjie Zhao
a
, Chao
Yi
a
, Longyan Wang
a
, Liwei Zhou
a
, Tingting Tang
a
, Qiufeng Yang
a
, Deshuai Lou
a, c
, Qiang
Li
a, c
, Yekuan Wu
a, c
, Linfeng Hu
a, c
, Gang Kuang
a
, Xi Liu
a
, Bochu Wang
b
#
, Minxuan Xu
a,
b, c #
a Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region,
School of Biological and Chemical Engineering, Chongqing University of Education,
Chongqing 400067, PR China
b Key Laboratory of Biorheological Science and Technology (Chongqing University),
Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030,
China.
c Research Center of Brain Intellectual Promotion and Development for Children Aged 0-6
Years, Chongqing University of Education, Chongqing 400067, PR China
# To whom correspondence should be addressed:
1. Prof. Jun Tan, Email: tanjun@cque.edu.cn; Chongqing Key Laboratory of Medicinal
Resources in the Three Gorges Reservoir Region, School of Biological and Chemical
Engineering, Chongqing University of Education, Chongqing 400067, PR China
2. Prof. Bochu Wang; Email: wangbc2000@126.com; Key Laboratory of Biorheological
Science and Technology (Chongqing University), Ministry of Education, College of
Bioengineering, Chongqing University, Chongqing 400030, China.
3. Dr. Minxuan Xu, Email: minxuanxu@foxmail.com; Chongqing Key Laboratory of
Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and
Chemical Engineering, Chongqing University of Education, Chongqing 400067, PR China
* These authors contributed equally to this work.
Abstract
Air pollution containing particulate matter (PM) less than 2.5 µm (PM
2.5
) plays an
essential role in regulating hepatic disease. However, its molecular mechanism is not
yet clear, lacking effective therapeutic strategies. In this study, we attempted to
investigate the effects and mechanisms of PM
2.5
exposure on hepatic injury by the in
vitro and in vivo experiments. At first, we found that PM
2.5
incubation led to a
significant reduction of nuclear factor erythroid-derived 2-related factor 2 (Nrf2),
along with markedly reduced expression of different anti-oxidants. Notably,
suppressor of IKKε (SIKE), known as a negative regulator of the interferon pathway,
was decreased in PM
2.5
-incubated cells, accompanied with increased activation of
TANK-binding kinase 1 (TBK1) and nuclear factor-κB (NF-κB). The in vitro studies
showed that Nrf2 positively regulated SIKE expression under the conditions with or
without PM
2.5
. After PM
2.5
treatment, Nrf2 knockdown further accelerated SIEK
decrease and TBK1/NF-κB activation, and opposite results were observed in cells
with Nrf2 over-expression. Subsequently, the gene loss- and gain-function analysis
demonstrated that SIKE deficiency further aggravated inflammation and TBK1/NF-
κB activation caused by PM
2.5
, which could be abrogated by SIKE over-expression.
Importantly, SIKE-alleviated inflammation was mainly dependent on TBK1
activation. The in vivo studies confirmed that SIKE- and Nrf2-knockout mice showed
significantly accelerated hepatic injury after long-term PM
2.5
exposure through
reducing inflammatory response and oxidative stress. Juglanin (Jug), mainly isolated
from Polygonum aviculare, exhibits anti-inflammatory and anti-oxidant effects. We
found that Jug could increase Nrf2 activation, and then up-regulated SIKE in cells and
liver tissues, mitigating PM
2.5
-induced liver injury. Together, all these data
demonstrated that Nrf2 might positively meditate SIKE to inhibit inflammatory and
oxidative damage, ameliorating PM
2.5
-induced liver injury. Jug could be considered as
an effective therapeutic strategy against this disease by improving Nrf2/SIKE
signaling pathway.
Keywords
PM
2.5
; Nrf2/SIKE; TBK1/NF-κB; inflammation and oxidative stress; Juglanin
1. Introduction
Particulate matter (PM) is a general term for all particulate matters in the atmosphere,
among which the type that has an aerodynamic diameter ≤ 2.5 µm (PM
2.5
) is the most
critical factor that affects human health [1,2]. The adverse effects of PM
2.5
are more
associated with PM
2.5
as a complex other than single or a just few components of
PM
2.5
particles [3]. The particle sizes, charges and combined effects of individual
components of PM
2.5
are all important for the adverse health impact of PM
2.5
exposure [4]. PM
2.5
exposure accounts for about 4.2 million deaths from lung cancer,
chronic lung disease, respiratory infections, heart disease and stroke, and is an
important ranking risk factor for death [5]. PM
2.5
can directly enter the circulatory
system, influencing the function of body tissues and organs, including liver [6,7].
Epidemiological study suggests that PM
2.5
pollution may be a risk factor for the
increase in the incidence of liver injury worldwide, and liver is the main organ for
metabolism and detoxification [8,9]. In rodent animals, the whole-body exposure to
concentrated ambient PM
2.5
results in a nonalcoholic steatohepatitis (NASH)-like
phenotype [10]. In the liver of mice with long-term exposure of PM
2.5
, the disruption
of hepatic lipid homeostasis, lobular and portal inflammation, and mild hepatic
steatosis have been identified [11]. Recently, we showed that prolonged PM
2.5
exposure promoted the risk of nonalcoholic fatty liver disease (NAFLD) through
elevating oxidative stress and inflammation [12]. Increasing evidence has
demonstrated that oxidative stress, inflammatory response, endoplasmic reticulum
(ER) stress and fibrosis are involved in the progression of PM
2.5
-induced tissue injury
[13-15]. Hepatic inflammation, as a pathological condition characterized by the
release of pro-inflammatory factors, occurs in most types of chronic liver diseases
such as NAFLD and hepatocellular carcinoma (HCC) [16,17]. Therefore, the studies
on PM
2.5
-induced hepatic inflammation are very important in terms of identifying new
health risk factors and understanding the pathogenesis of liver diseases. However, the
present studies regarding the effects of PM
2.5
on liver damage or inflammation are
limited, requiring further exploration.
Nrf2 is the master transcriptional regulator of antioxidant gene expression. Upon
increased oxidative stress, an adaptor for Nrf2 degradation, known as Kelch-like ECH
associated protein 1 (Keap1), is directly meditated by oxidants in the cytoplasm,
leading to stabilization and activation of Nrf2 [18]. Nrf2 modulates more than 200
genes encoding cytoprotective phase II detoxification and anti-oxidant enzymes, such
as heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone 1) (NQO-1),
glutamate-cysteine ligase subunits (GCLC and GCLM) and glutathione-S-transferase
(GST) [19]. Chronic elevation of reactive oxygen species (ROS) is a pivotal factor
that results in PM
2.5
-elicited tissue injury [15,20]. Our previous studies indicated that
PM
2.5
-induced hypothalamus inflammation, renal injury and cardiomyopathy were
closely associated with the deregulation of Nrf2 signaling, subsequently regulating
inflammatory response both in vivo and in vitro [15,21,22]. Nrf2-regulated signaling
pathway was recently suggested to modulate PM-induced hepatic insulin resistance
[23]. Moreover, Nrf2 could meditate the expression of numerous anti-inflammatory
genes by antioxidant response elements in their promoters to neutralize free radicals,
and enhance removal of environmental toxins [24,25]. With the understanding of the
mechanistic knowledge of the Keap1-Nrf2 system, a number of Keap1-Nrf2 PPI
inhibitors have been developed as promising therapeutics against chronic and
inflammatory conditions [26-28]. Unfortunately, the therapeutic potential of the
Keap1-Nrf2 on inflammatory hepatic disease induced by air pollution remains unclear.
Suppressor of IKKε (SIKE), as a small coiled-coil domain-containing protein
comprised of 207 amino-acid residues, is widely expressed in most tissues, such as
the heart, stomach and kidney [29]. SIKE has been identified as an interaction partner
for IKKε and a negative modulator of the interferon pathway through negatively
regulating TBK1-involved signaling [30]. Additionally, SIKE might be an important
negative regulator of pathological cardiac hypertrophy via interacting with TBK1,
contributing to cardiac remodeling [31]. Given the critical role of TBK1 in promoting
inflammatory response [32], we hypothesized that SIKE/TBK1 might be involved in
inflammatory disease. Although the immune response meditated by SIKE has been
indicated, its additional biological activities are still far from to be investigated,
especially its association with oxidative stress regulated by Nrf2.
Growing studies have demonstrated that a large number of antioxidants exhibit
promising activities to reduce oxidative stress and inflammatory response during the
progression of acute or chronic hepatic injury [33,34]. Juglanin (Jug), a natural
compound belonging to flavonoids, is isolated from crude Polygonum aviculare. Jug
exerts anti-inflammatory, anti-fibrotic and anti-cancer effects [35-37]. Jug suppresses
IL-1β-induced inflammation in human chondrocytes [35]. In lipopolysaccharide
(LPS)-induced acute lung injury, Jug dramatically reduced the inflammation of cell
infiltration and NF-κB activation [38]. It also regulates ROS production to inhibit
breast cancer progression [36]. More recently, Jug was reported to restrain the
Stimulator of interferon genes (STING), suppressing inflammation to protect against
bleomycin-induced lung injury [39].
According to these previous findings, we hypothesized that Nrf2- and SIKE-
associated signaling pathways might be involved in PM
2.5
-induced hepatic injury
through regulating oxidative stress and inflammatory response. On the other, Jug was
subjected to animals and cells challenged with PM
2.5
to explore whether it could be a
promising therapeutic to alleviate air pollution-induced liver disease by targeting Nrf2
and SIKE signaling.
2. Materials and methods
2.1. PM
2.5
sampling preparation
The method for PM
2.5
sampling preparation was based on previous studies with little
modification [40,41]. In brief, to collect exposure mass, quartz filter (8 cm × 10 cm,
2500QATUP, Pallflex Products, Putnam, CT, USA) was used to continuously and
weekly gather PM
2.5
from Yuquan Road, Beijing, China (January-June 2016) at a
flow rate of 166 L/min. Ambient PM
2.5
filters were then maintained in -80°C until
administration. Then, the sampling was treated with anhydrous alcohol and dissolved
in pyrogen-free water. Subsequently, the extraction was sonicated for 48 h in
ultrasonic box and concentrated through vacuum freeze-drying. Next, double-distilled
water was added to freeze-dried product, which was centrifuged at 5000 rpm. The
water-insoluble fraction was suspended in D-Hank’s buffer (Gibco Corporation, USA)
and vortexed prior to further analysis. The components of PM
2.5
were shown in
Supplementary table 1.
2.2. Cell culture and treatment
2.2.1. Cell culture
The HEK-293 cell line was purchased from InvivoGen (InvivoGen Code: hkb-
tnfdmyd). Human liver cell line L02 was obtained from Procell Life Science &
Technology Co.,Ltd. (Shanghai, China). All cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal
bovine serum (FBS, Gibco), 100 U/ml of penicillin, and 100 μg/ml streptomycin at
37°C and 5% CO
2
. Juglanin (HPLC purity ≥98%) and Nrf2 activator tBHQ (purity
≥98%) were purchased from Shanghai YuanMu Biological Technology Co. Ltd.
(Shanghai, China) and Sigma-Aldrich (USA), respectively.
2.2.2. Cell transfection
The commercial human Nrf2 RNAi products were obtained from Novus Biologicals
(H00004780-R01) and used as indicated in product specification. The negative
control (siNC) and Nrf2-specific siRNAs (siNrf2) were synthesized and obtained
from Shanghai Generay Biotech (Shanghai, China). A cDNA encoding human Nrf2
was generated by PCR from human brain cDNA library (Invitrogen, USA) and
subcloned into the eukaryotic expression plasmid pcDNA3-Flag. And the empty
vector plasmid (EP) was served as control group. The p-NF-κB-luciferase reporter
plasmid was purchased from Beyotime Institute of Biotechnology (Shanghai, China).
Human SIKE and p-TBK1 luciferase reporter plasmids based on pcDNA3.1 vector, as
well as mammalian expression plasmids encoding HA-tagged SIKE or control
plasmid based on pCMV-C-HA plasmid (Beyotime Institute of Biotechnology) were
constructed by standard molecular biology techniques. Lipofectamine®3000
(Invitrogen) was used for cell transfection following the manufacturer’s protocols.
2.2.3. Recombinant adenovirus generation
To overexpress SIKE, the entire coding region of the human SIKE gene, under
control of the cytomegalovirus promoter, was encompassed by replication-defective
adenoviral vectors. To knockdown SIKE expression, three human shSIKE constructs
were obtained from Santa Cruz (sc-88645). The construct that decreased SIKE levels
to the greatest extent was selected for all further analysis. We also constructed
constitutively active TBK1 (Ad-TBK1) and shTBK1 (Ad-shTBK1). A similar
adenoviral vector encoding the green fluorescent protein (GFP) gene was used as a
control, and short hairpin RNA (Ad-shRNA) served as controls. L02 cells were
infected with adenovirus in diluted media at a multiplicity of infection of 100 for 24 h.
2.3. Luciferase detection
HEK-293 cells (2 × 10
5
/ml) were plated in 24-well dish and transfected using
Lipofectamine 3000 (Invitrogen), with plasmid encoding p-NF-κB, p-TBK1 and
SIKE luciferase reporter (firefly luciferase; 100 ng) and pRL-TK (renilla luciferase
plasmid; 10 ng) together with siNrf2 or Nrf2 plasmids. Empty pcDNA3.1 vector was
used to maintain equal amounts of DNA among wells. Cells were incubated with 0,
25, 50, and 100 μg/ml of PM
2.5
after 24 h transfection. Then, the cells were collected
and subjected to luciferase activity analysis using a Dual-luciferase Assay (Promega,
USA) with a Luminoskan Ascent luminometer (Thermo Fisher Scientific, USA)
according to the manufacturer’s protocols. Reporter gene activity was analyzed by
normalization of the firefly luciferase activity to renilla luciferase activity.
2.4. Western blotting
For nuclear protein extraction in cells, nuclear and Cytoplasmic Protein Extraction Kit
(Beyotime, Nanjing, China) was used according to the manufacturer’s instructions.
For extraction of total protein samples, cells and hepatic tissues were homogenized
using 10% (wt/vol) hypotonic buffer (pH 8.0, 5 μg/ml soybean trypsin inhibitor, 1
mM EDTA, 4 mM benzamidine, 1 mM Pefabloc SC, 25 mM Tris-HCl, 5 μg/ml
leupeptin, 50 μg/ml aprotinin) to yield a homogenate. The final supernatants were
collected via centrifugation at 12,000 rpm for 15 min at 4°C. Bicinchoninic acid
(BCA) protein analysis kit (Thermo Fisher Scientific, USA) was used for the
measurements of protein concentration according to its instructions. Then, 40 μg
protein was subjected to 10-12% SDS-Polyacrylamide-Gel-Electrophoresis (SDS-
PAGE) and transferred to polyvinyldene fluoride (PVDF) membranes (Millipore,
USA). After blocking in 5% skim milk, all membranes were incubated with primary
antibodies (Supplementary table 2) overnight at 4°C. Then, the membranes were
incubated with corresponding secondary antibodies (Supplementary table 2) for 1 h at
room temperature. The signal was detected with the enhanced chemiluminescence
(ECL) Detection system (Thermo Fisher Scientific). Each protein expression was
analyzed using Image Lab Software (Version 1.4.2b, National Institutes of Health,
USA) and normalized to GAPDH.
2.5. Quantitative real-time PCR (RT-qPCR)
Trizol reagent (Invitrogen, USA) was used to extract the total RNA samples from
cells or hepatic tissues according to the manufacturer’s introductions. Specifically, 1
μg of total RNA extraction was reverse transcribed with the M-MLV-RT system
(Promega, Shanghai, China) following the manufacturer’s protocols. The program
was performed at 42°C for 1 h and terminated through deactivation of the enzyme at
70°C for 10 min. Subsequently, PCR was performed with SYBR Green (Bio-Rad,
USA) on an ABI PRISM 7900HT detection system (Applied Biosystems, USA). The
primer sequences (Supplementary table 3) used in this study were produced by
Invitrogen Corporation or Generay Biotech (Shanghai, China). The quantification
analysis was performed following the 2
-ΔΔCt
expressions. Relative fold expression of
every target gene expression was normalized to GAPDH.
2.6. Cell viability
The MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China)
was used to determine various cells viability according to the manufacturer
instructions. The absorbance was finally detected using a microplate reader at 570 nm.
2.7. ROS production in vitro
After various treatments, 1.5 mL of dichloro-dihydro-fluorescein diacetate (DCFH-
DA) (10.0 μM, Sigma-Aldrich) was added to cells at 37°C for 25 min. Then, the
samples were analyzed with a fluorescence microscopy (Olympus, Tokyo, Japan).
2.8. Animals and treatments
2.8.1. Ethical approval and animals
All animal experiment protocols were approved by the Institutional Animal Care and
Use Committee in Chongqing Key Laboratory of Medicinal Resources in the Three
Gorges Reservoir Region, School of Biological and Chemical Engineering,
Chongqing University of Education (Chongqing, China). The methods used in this
research were in accordance with the Regulations of Experimental Animal
Administration issued by the Ministry of Science and Technology of the People’s
Republic of China. 1) The wild type male mice with C57BL/6 background (6-8 weeks
old, weighing 20 ± 2 g) were purchased from Beijing Vital River Laboratory Animal
Technology Co., Ltd. (Beijing, China). 2) Nrf2 knockout C57BL/6J mice (Nrf2
-/-
)
were purchased from the Jackson Laboratory (Bar Harbor, ME). 3) SIKE knockout
C57BL/6 mice (SIKE
-/-
) were created by Cyagen Biosciences Inc. (Suzhou, China) by
CRISPR/Cas-mediated genome engineering. All pups will be genotyped by PCR
followed by sequencing analysis. All animals were maintained in a specific pathogen-
free (SPF) facility with constant temperature and humidity under a 12 h dark/light
cycle, and free access to food and water.
2.8.2. Mice whole body exposure and Juglanin treatment
We established the “real-ambient exposure” system optimizing the inhabited
environment, which ensured the availability of food and water for mice and whole-
body inhalation PM exposure to mice at the same time. All male mice (6-8 weeks of
old, weighing 20 ± 2 g) were exposed to concentrated PM
2.5
(150.1 ± 2.5 μg/m
3
, flow
rate of 70 L/min; moderate pollution and equal to air quality rating-4 level [115-150
μg/m
3
] based on China environmental pollution standards) or filtered air (FA, served
as control) for 6 h/day, 5 times a week in a mobile exposure system-HOPE-MED
8052 automatic nose and mouth type inhalation exposure system (Hepu Industry and
Trade Co., Ltd., China) according to previous studies [15,21,22]. The wild type mice
were simultaneously treated with Juglanin (40 mg/kg) once daily via gavage for 6 h
prior to PM
2.5
or FA exposure. After PM
2.5
challenge for 24 weeks with or without
Juglanin administration, all mice were sacrificed for eye blood collection. Body
weight of mice was measured each week. Blood pressure of each mouse was weekly
measured using a noninvasive blood pressure meter (Surgivet, USA). The hepatic
tissue samples were isolated from mice for further analysis.
2.9. Biochemical analysis in vitro and in vivo
Serum levels of tumor necrosis factor-α (TNF-α) (#MTA00B), interleukin-1β (IL-1β)
(#MLB00C), IL-6 (#M6000B) and interferon-β (IFN-β) (#MIFNB0) were measured
using enzyme-linked immunosorbent assay (ELISA) commercial kits (R&D System,
USA) according to the manufacturer’s instructions. Superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx) and malondialdehyde (MDA) levels in
cells or liver samples were measured using commercial kits obtained from Nanjing
Jiancheng Bioengineering Institute (Nanjing) according to the protocols
recommended by the manufacturer. Aspartate transaminase (AST) and alanine
transaminase (ALT) in hepatic samples were assessed with commercial kits (Nanjing
Jiancheng Bioengineering Institute) according to the manufacturer’s protocols.
2.10. Histochemical analysis
The mice liver tissues were fixed with 10% neutral formalin, embedded in paraffin,
and sectioned transversely. Thin sections (≤ 15 μm) were stained with hematoxylin
and eosin (H&E) following the standard histopathological processes. All sections
were detected by 3 histologists without knowledge of the treatment procedure.
2.11. Immunofluorescence
After each treatment, the cells were washed with PBS, fixed in 4% paraformaldehyde
for 30 min at room temperature, blocked with 5% (w/v) bovine serum albumin (BSA,
Sigma Aldrich, USA) in PBST, and immunostained with anti-Nrf2 (1:100 dilution;
Abcam, USA), anti-SIKE (1:100 dilution; Thermo Fisher Scientific, USA), anti-NF-
κB (1:100 dilution; Abcam) and anti-p-TBK1 (1:100 dilution; Cell Signaling
Technology, USA) antibody overnight at 4°C, followed by incubation with a goat
anti-mouse Alexa Fluor-488-conjugated secondary antibody and/or goat anti-rabbit
Alexa Fluor-594-conjugated secondary antibody (Abcam). After washing with PBS,
the cells were stained with DAPI (Beyotime, Shanghai, China) and observed under a
fluorescence microscope. Immunofluorescence staining for liver sections was
performed using anti-Nrf2 (1:100 dilution; Abcam) and anti-SIKE (1:100 dilution;
Thermo Fisher Scientific) as described for cells with little modification.
Immunofluorescence images were obtained using a fluorescence microscope. Images
were analyzed with Image J software.
2.12. Chromatin immunoprecipitation (ChIP) assay
Cells were stimulated with or without PM
2.5
. The corresponding control cells were
cultured to 80-90% confluency. Then, ChIP analysis was conducted using the
Enzymatic Chromatin IP Kit (Millipore) according to the manufacturer’s protocols.
The antibodies as follows were used to immuno-precipitate crosslinked protein-DNA
complexes: rabbit anti-Nrf2 and normal rabbit IgG. The immunoprecipitated DNA
was purified for PCR assay with the primers that were specific for the putative
binding sites within the promoter of SIKE.
2.13. Statistical analysis
Data were expressed as the means ± standard error (SEM). GraphPad PRISM (version
6.0; GraphPad Software, USA) was used for data analysis. Unpaired Student’s t-test
was performed to calculate the difference between two groups, and one-way analysis
of variance (ANOVA) followed by Bonferroni’s post hoc test was used for calculation
between multiple experimental groups. The Pearson single correlation analysis was
used to determine the correlation between the expression of cellular p-TBK1 positive
cells and nuclear NF-κB positive cells. Values of P < 0.05 were considered
statistically significant. Animal feeding and treatment, and histological analysis were
performed in a single-blinded fashion.
3. Results
3.1. PM
2.5
incubation inhibits Nrf2 and SIKE activation in vitro
At first, we attempted to explore if Nrf2, as well as associated signaling, could be a
potential factor during the pathogenesis of liver related diseases. At first, CCK-8
analysis was used to calculate the cell viability in L01 cells treated with or without
PM
2.5
at different concentrations. We found that PM
2.5
exposure from 25 μg/ml
significantly caused the reduction of the cell viability in L02 (Supplementary fig. 1A).
RT-qPCR and western blotting results demonstrated that PM
2.5
at lower concentration
slightly up-regulated Nrf2 expression though no significant difference was detected.
Then, we found that PM
2.5
incubation dose-dependently reduced the expression of
Nrf2 in human liver cell line L02 (Fig. 1A). Similar results were observed in the
expression change of anti-oxidants including HO-1, NQO-1, GCLC and GCLM in
PM
2.5
-exposed L02 cells (Fig. 1B). Furthermore, Nrf-2 expression both from mRNA
and protein levels was time-dependently decreased by PM
2.5
incubation (Fig. 1C).
Also, HO-1, NQO-1, GCLC and GCLM mRNA expression levels were markedly
reduced by PM
2.5
treatment in a time-dependent manner (Fig. 1D). Subsequently, we
found that SIKE expression levels were significantly down-regulated by PM
2.5
in a
dose- and time-dependent manner. In contrast, p-TBK1 and p-NF-κB protein
expression levels were greatly up-regulated in L02 cells in response to PM
2.5
(Fig. 1E-
H). Therefore, results above demonstrated that PM
2.5
exposure reduced Nrf2 and
SIKE expression, while promoted the activation of TBK1 and NF-κB in liver cells.
Because 25, 50 and 100 μg/ml of PM
2.5
showed the significant influence on the cell
viability, Nrf2 and SIKE expression change, and thus were selected for the subsequent
in vitro experiments.
3.2. Nrf2 positively regulates SIKE expression in PM
2.5
-incubated cells
Subsequently, Nrf2 was inhibited or over-expressed to further explore its regulatory
effect on SIKE and TBK1/NF-κB signaling. As shown in Fig. 2A, Nrf2 was
successfully inhibited by transfection with its specific siRNA. As expected, HO-1,
NQO-1, GCLC and GCLM expression levels were markedly reduced in L02 cells
with Nrf2 knockdown (Fig. 2B). Notably, western blot and RT-qPCR results showed
that SIKE protein and mRNA expression levels were greatly decreased in L02 cells
transfected with siNrf2 (Fig. 2C). HEK-293 cells have been widely used for studying
gene function due to its relatively higher transfection efficiency [42]. To further study
the relation between SIKE signaling and Nrf2 activation with greater depth, we
transfected HEK-293 cells with luciferase reporter vectors and/or siNrf2, followed by
treatment with 0, 25, 50 and 100 μg/ml of PM
2.5
for another 24 h. As shown in Fig.
2D, knockdown of Nrf2 significantly down-regulated SIKE, while promoted p-TBK1
and p-NF-κB activation reporter gene expression, compared with normal cells. Then,
Nrf2 was over-expressed in L02 cells by transfection with its specific plasmids, which
were along with greatly increased expression of HO-1, NQO-1, GCLC and GCLM
(Fig. 2E and F). Of note, SIKE expression both from protein and mRNA levels were
highly elevated in L02 cells with Nrf2 over-expression (Fig. 2G). Luciferase reporter
analysis also demonstrated that Nrf2 over-expression abrogated the regulatory effects
of PM
2.5
on SIKE, p-TBK1 and p-NF-κB activation in HEK-293 cells (Fig. 2H).
Results above demonstrated that Nrf2 might positively regulate SIKE expression,
whereas inhibit TBK1 and NF-κB activation. Immunofluorescence staining further
confirmed that PM
2.5
stimulation obviously reduced Nrf2 and SIKE expression levels,
as evidenced by the weaker fluorescence (Fig. 2I). Furthermore, ChIP assay detected
the binding of Nrf2 on the SIKE promoter region in L02 cells with or without PM
2.5
exposure (Fig. 2J). Subsequently, western blot analysis confirmed that Nrf2
knockdown markedly reduced SIKE expression under normal condition. PM
2.5
-
induced increases in p-TBK1 and p-NF-κB were significantly further elevated by
Nrf2 knockdown (Fig. 2K). In contrast, SIKE expression was up-regulated when Nrf2
was over-expressed in L02 cells in the absence of PM
2.5
. In response to PM
2.5
,
promoting Nrf2 could greatly rescued SIKE expression in L02 cells, whereas
effectively down-regulated p-TBK1 and p-NF-κB (Fig. 2L). Therefore, results above
indicated that Nrf2 might positively regulate SIKE, contributing to the blockage of
TBK1 and NF-κB.
3.3. Effects of SIKE on TBK1/NF-κB signaling pathway in PM
2.5
-treated cells
TBK1/NF-κB signaling pathway contributed to inflammatory response [32,43,44]. To
further explore the underlying molecular mechanism, the gain- and loss-of-function
studies were conducted in L02 cells through infection with either Ad-shSIKE to
suppress SIKE expression or Ad-SIKE to over-express SIKE (Fig. 3A). The cells
were subsequently treated with PM
2.5
for 24 h. Results showed that SIKE deletion
markedly promoted the expression of inflammatory factors including IL-1β, IL-6,
TNF-α and IFN-β induced by PM
2.5
, while SIKE over-expression effectively reduced
the expression of these factors (Fig. 3B). Then, western blotting showed that SIKE
knockdown further accelerated the activation of TBK1 and NF-κB in PM
2.5
-incubated
L02 cells, whereas over-expressing SIEK significantly reduced the phosphorylation of
TBK1 and NF-κB (Fig. 3C). NF-κB nuclear expression is critical for the release of
pro-inflammatory regulators, and was thus investigated in L02 cells. Western blotting
results showed that nuclear NF-κB expression levels were markedly elevated by PM
2.5
stimulation, while being further promoted in cells with SIKE knockdown. As
expected, SIKE over-expression significantly reduced NF-κB nuclear translocation
(Fig. 3D). Immunofluorescence staining indicated that NF-κB nuclear translocation
stimulated by PM
2.5
was further enhanced by SIKE deficiency, along with obviously
up-regulated expression of p-TBK1. In contrast, improving SIKE expression
restrained NF-κB nuclear translocation in PM
2.5
-treated cells, and decreased p-TBK1
expression levels were detected, as evidenced by the weaker fluorescence (Fig. 3E).
Western blot analysis further suggested that the inhibitory effects of SIKE on TBK1
phosphorylation were dose-dependent (Supplementary fig. 1B). Analysis of all groups
as a single cohort found a significant correlation between the intensity of p-TBK1
positive cells in cells with the intensity of NF-κB positive cells in nuclear
(Supplementary fig. 1C). These data implicated SIKE as a negative regulator of
hepatic injury through suppressing the activation of TBK1/NF-κB signaling.
3.4. SIKE-regulated inflammatory response requires TBK1 blockage in PM
2.5
-treated
cells
To further explore if SIKE-regulated inflammatory response was TBK1-dependent,
we constructed adenoviral vectors to knock down TBK1 (Ad-shTBK1) or over-
express TBK1 (Ad-TBK1) in L02 cells (Supplementary fig. 2). Under basal
conditions, alterations in either SIKE or TBK1 expression had no significant effect on
the expression of inflammatory factors. When L02 cells were exposed to PM
2.5
,
however, co-infection with Ad-TBK1 counteracted Ad-SIKE-reduced expression of
IL-1β, IL-6, TNF-α and IFN-β, as well as p-NF-κB (Fig. 4A and B). Conversely, Ad-
shSIKE-promoted expression of pro-inflammatory factors and p-NF-κB were
markedly abolished by Ad-shTBK1 (Fig. 4C and D). Thus, TBK1 was indispensable
for SIKE-regulated inflammation.
3.5. Juglanin improves Nrf2 activation to inhibit oxidative stress in PM
2.5
-incubated
cells
Juglanin was then chosen to further explore if pharmacological intervention could
alleviate PM
2.5
-induced ROS production and inflammation by improving Nrf2/SIKE
signaling pathway. As shown in Fig. 5A and B, Jug treatments at different
concentrations had no significant cytotoxicity to L02 cells. In addition, 40 μM of Jug
treatment showed to cytotoxic effects on L02 cells. CCK-8 confirmed that the
viability of cells treated with Jug alone was similar to that of the control group
(Supplementary fig. 3A). As presented in Supplementary fig. 3B, the cellular LDH
release was not markedly different between the control group and the group treated
with Jug alone. Western blotting and immunofluorescence staining confirmed that Jug
treatments markedly induced Nrf2 nuclear translocation in a dose-dependent manner
(Fig. 5C and D). In contrast, Keap-1, as an essential suppressor of Nrf2, was down-
regulated by Jug in PM
2.5
-exposed cells, which was similar with the effects of tBHQ,
known as a typical Nrf2 activator. In addition, HO-1 expression levels were slightly
elevated by Jug under normal conditions. Also, PM
2.5
-decreased HO-1 expression was
markedly restored by Jug co-treatment (Fig. 5E). Furthermore, in cells without PM
2.5
stimulation, SOD, CAT and GPX activities were markedly improved by Jug treatment.
Also, PM
2.5
-reduced SOD, CAT and GPX levels were highly rescued in L02 cells co-
treated with Jug (Fig. 5F). Finally, DCF-DA staining showed that Jug treatment could
evidently reduce ROS accumulation in PM
2.5
-stimulated L02 cells (Fig. 5G).
Therefore, these data demonstrated that Jug exhibited anti-oxidative effects to
decrease PM
2.5
-caused ROS accumulation mainly through activating Nrf2.
3.6. Juglanin treatment suppresses inflammation in PM
2.5
-incubated cells through
increasing SIKE expression
In this regard, we found that Jug treatment caused slight up-regulation of SIEK in L02
cells without PM
2.5
stimulation. As expected, PM
2.5
-induced cells had lower SIKE
expression compared with the control group, which was, however, markedly
improved by Jug co-treatment (Fig. 6A). Immunofluorescence staining confirmed that
Jug administration could rescue SIKE expression in PM
2.5
-stimulated cells, as
evidenced by the stronger fluorescence than that of the PM
2.5
-alone group (Fig. 6B).
Then, RT-qPCR analysis showed that Jug incubation markedly decreased the mRNA
levels of IL-1β, IL-6, TNF-α and IFN-β in L02 cells with PM
2.5
stimulation (Fig. 6C).
Consistently, PM
2.5
-promoted p-TBK1 and p-NF-κB were significantly abolished by
Jug co-incubation (Fig. 6D). Therefore, results above illustrated that Jug had anti-
inflammatory effects to alleviate PM
2.5
-induced injury in vitro, which might be
associated with the improvement of SIEK signaling.
3.7. Effects of SIKE expression on PM
2.5
-induced hepatic injury in mice
The in vitro studies showed that Nrf2/SIKE signaling might have protective effects
against PM
2.5
-induced oxidative and inflammatory damage by improving anti-
oxidants and suppressing TBK1/NF-κB signaling. To further elucidate in vivo effect
of PM
2.5
exposure, as well as underlying molecular mechanisms, male C57BL/6 mice
were exposed to concentrate ambient PM
2.5
for 24 weeks. To evaluate the intracellular
impact of PM
2.5
exposure, we performed transmission electron microscopy (TEM)
analysis of the cell ultrastructure with the liver tissue samples from the mice exposed
to PM
2.5
or FA. The liver of the mice exposed to PM
2.5
displayed ultrastructure
damage when compared with those from the mice exposed to FA (Supplementary fig.
4A). Our preliminary experiment showed that PM
2.5
long-term exposure caused
decreases in SIKE in liver tissues of wild type mice (Supplementary fig. 4B). To
further confirm these findings, the in vivo studies were performed using SIKE
+/+
or
SIKE
-/-
mice with or without PM
2.5
challenge. SIKE was not detectable in liver of
SIKE
-/-
mice (Supplementary fig. 4C). As shown in Fig. 7A, long-term PM
2.5
exposure showed no significant effects on the change of body weight in both wild
type and SIKE-knockout mice. Nevertheless, PM
2.5
exposure led to a significant
increase in MBP compared with the FA group; however, SIKE deletion could further
promote MBP in PM
2.5
-challenged mice (Fig. 7B). H&E staining showed that PM
2.5
exposure resulted in histological changes in liver sections, which were further
accelerated by SIKE deletion (Fig. 7C). Hepatic function in mice was then assessed.
As expected, PM
2.5
-challenged mice had higher AST and ALT in liver than that of the
FA mice, which were, however, further aggravated by SIKE deficiency
(Supplementary fig. 5A). In addition, PM
2.5
caused a reduction in liver weight of mice
compared with the FA mice. SIKE deletion showed no significant effects on the
change of liver weight in PM
2.5
-exposed mice (Fig. 7D). Subsequently, in response to
PM
2.5
, significantly increased IL-1β, IL-6 and TNF-αwere detected in liver samples,
which were further promoted in mice with SIKE deficiency (Fig. 7E). RT-qPCR
analysis further confirmed that compared to the PM
2.5
-single group, SIKE absence led
to higher expression of IL-1β, IL-6, TNF-α and IFN-β in hepatic tissues of PM
2.5
-
treated mice, accompanied with markedly up-regulated activation of TBK1 and NF-
κB (Fig. 7F and G). Subsequently, we unexpectedly found that PM
2.5
-inhibited
expression of anti-oxidants such as HO-1, GCLC and GCLM were further down-
regulated in mice with SIKE deletion (Fig. 7H). Moreover, after PM
2.5
challenge,
SIKE
-/-
mice showed lower SOD activity and higher MDA levels in hepatic samples
(Fig. 7I). These data demonstrated that SIKE deletion could further accelerate PM
2.5
-
induced hepatic injury and dysfunction by promoting inflammatory response and
oxidative stress.
3.8. Effects of Nrf2 activation on PM
2.5
-induced hepatic injury in mice
Here, mice with or without Nrf2 expression were included to further confirm our
hypothesis. At first, western blot results demonstrated that Nrf2 was not detectable in
hepatic tissues of Nrf2
-/-
mice (Supplementary fig. 6). Consistently, PM
2.5
long-term
exposure showed no significant effects on the change of body weight either in Nrf2
+/+
or Nrf2
-/-
mice (Fig. 8A). Also, MBP was clearly increased in PM
2.5
-challenged mice,
which were further enhanced in mice without Nrf2 expression (Fig. 8B). PM
2.5
caused
a significant decrease in the liver weight of mice, and Nrf2 knockout showed no
significant effects on the change of liver weight (Fig. 8C). PM
2.5
-induced histological
alterations were further enhanced in mice with Nrf2 knockout, accompanied with
markedly promoted hepatic ALT and AST levels (Fig. 8D and Supplementary fig. 5B).
Thus, Nrf2 was critically involved in the maintaining of hepatic function in mice
when stimulated by PM
2.5
. We then found that PM
2.5
-reduced hepatic SOD activity
was further decreased in Nrf2
-/-
mice, while MDA levels in liver were greatly up-
regulated (Fig. 8E). Compared with the PM
2.5
-treated mice, Nrf2
-/-
mice showed lower
HO-1, NQO-1, GCLC and GCLM expression levels in hepatic tissues (Fig. 8F).
Subsequently, we found that Nrf2
-/-
mice without PM
2.5
exposure had lower SIKE
expression levels than that of the wild type group, which were in line with the results
observed in vitro. However, after long-term PM
2.5
challenge, Nrf2
-/-
mice showed
further reduced expression of hepatic SIKE compared to the PM
2.5
-alone group (Fig.
8G-I). RT-qPCR analysis confirmed that after PM
2.5
exposure for 24 weeks, Nrf2
deletion markedly enhanced the expression of pro-inflammatory factors, including IL-
1β, IL-6, TNF-α and IFN-β, accompanied with accelerated activation of TBK1 and
NF-κB (Fig. 8J and K). Therefore, these in vivo findings elucidated that Nrf2 could
regulate SIKE expression to protect against PM
2.5
-inudced liver damage.
3.9. Juglanin ameliorates PM
2.5
-induced hepatic injury through improving Nrf2/SIKE
signaling pathway in mice
Here, we attempted to further explore the potential of Jug to improve PM
2.5
-inudced
hepatic injury in vivo. Consistently, there was no significant difference in the change
of body weight from each group of mice (Fig. 9A). PM
2.5
-induced MBP was
effectively reduced by Jug co-treatment (Fig. 9B). PM
-2.5
-reduced liver weight was
rescued in Jug-treated mice (Fig. 9C). Jug administration also improved PM
2.5
-
induced hepatic dysfunction, as evidenced by the markedly reduced hepatic ALT and
AST levels (Supplementary fig. 5C). H&E results confirmed that Jug supplementation
alleviated the histological impairments in hepatic samples of mice challenged with
PM
2.5
. Immunofluorescence staining showed that Nrf2 and SIKE expression were
slightly increased by Jug administration, which were both evidently reduced in liver
of PM
2.5
-challenged mice. However, Jug co-treatment clearly improved Nrf2 and
SIKE expression in PM
2.5
-treated mice (Fig. 9D). Western blot analysis further
demonstrated that PM
2.5
-decreased Nrf2 and SIKE could be restored by Jug co-
treatment, while p-TBK1 and p-NF-κB expression levels stimulated by PM
2.5
were
effectively repressed in mice with Jug intervention (Fig. 9E). As shown in Fig. 9F,
PM
2.5
exposure markedly reduced Nrf2 expression from mRNA levels, which were
rescued in mice co-treated with Jug. Compared with the PM
2.5
group, hepatic SOD
activities were markedly rescued by Jug. In contrast, PM
2.5
-enhanced MDA levels
were markedly abrogated by Jug administration (Fig. 9G). Finally, RT-qPCR analysis
showed that PM
2.5
-induced oxidative stress and inflammatory response were
effectively diminished by Jug co-treatment, as evidenced by the rescued expression of
HO-1, NQO-1, GCLC and GCLM, and the reduced mRNA levels of IL-1β, IL-6,
TNF-α and IFN-β (Fig. 9H). Taken together, these data suggested that Jug could
alleviate PM
2.5
-induced liver injury by improving Nrf2/SIKE signaling to suppress
oxidative stress and inflammation.
4. Discussion
According to the statistics of World Health Organization (WHO), the outdoors
pollution amongst the top 8 environmental risks to health. Especially in China and
India, adding to the challenge are the side effects of rapid economic development: air
pollution, contaminated water, and encroaching urbanization, all of which threaten
health of people [45,46]. Toxicological studies have indicated that the toxicity of
PM
2.5
is a result of the combined effect of particles and the adsorbed toxic pollutants
[3,4]. The harmful effect triggered by PM includes the oxidative stress mechanism,
which can regulate the pathogenesis of inflammation in various organs [15,22,47,48].
The signaling pathways through which PM
2.5
exposure promotes hepatic dysfunction
and impairments of hepatic lipid/glucose metabolism have been widely revealed [8-
10,23]. However, the pronounced effect of PM
2.5
exposure on the meditation of
hepatic pathways associated with oxidative stress and inflammation has not been fully
characterized.
The composition of PM
2.5
is very complex, and the organic carbon particles and
insoluble heavy metals may find their way to the systemic circulatory system and
extrapulmonary organs from their deposition sites [49,50]. Therefore, these toxic
compositions may have extensive and rapid adverse reactions in the liver. In this
study, the top of ambient PM
2.5
among all chemical components was Sulfur, and
Sulfur was the principle component and associated with PM
2.5
-induced disease
[51,52]. PM
2.5
concentration at 150.1 ± 2.5 μg/m
3
that’s equal to air quality rating-4
level in China standard was used for in vivo experiments to mimic the real-world
ambient PM
2.5
for individuals. Thus, the exposure concentrations of PM
2.5
in the
exposure chamber were much higher than the WHO Air Quality Guideline in 2005
goals with PM
2.5
at 25 mg/m
3
(~6.0-fold of the WHO goal for PM
2.5
). Moreover, we
clearly indicated that ambient PM
2.5
exposure induced oxidative stress and
inflammatory response in liver of mice through restraining Nrf2 and SIKE activation,
leading to hepatic injury consequently. There was an interaction between oxidative
stress and inflammation caused by PM
2.5
exposure. A critical finding in this study was
that Nrf2 might positively modulate SIKE expression with or without PM
2.5
stimulation. Nrf2 deficiency promoted SIKE reduction, subsequently enhancing
TBK1/NF-κB activation and associated inflammatory response. However, Nrf2 over-
expression increased SIKE expression and decreased the release of inflammatory
factors, along with the inactivation of TBK1/NF-κB. Moreover, the in vitro studies
showed that SIKE negatively regulated NF-κB activation to control the pro-
inflammatory response in PM
2.5
-incubated liver cells, which was largely dependent on
TBK1 inactivation. The in vivo experiments using loss-of-function approaches
confirmed that Nrf2/SIKE signaling exhibited protective effects against PM
2.5
-
induced hepatic injury by mitigating oxidative stress and inflammation. The
pharmacological intervention analysis showed that Jug treatment could activate Nrf2
signaling, and then improve SIKE, subsequently inhibiting oxidative and
inflammatory damage induced by PM
2.5
. Collectively, all these findings demonstrated
that Nrf2 may positively regulate SIKE expression to suppress oxidative stress and
inflammation, ameliorating PM
2.5
-triggered hepatic damage. Jug, as a potential Nrf2
activator, could attenuate hepatic oxidative stress and inflammatory response induced
by PM
2.5
through improving SIKE.
Nrf2 is the predominant manager of the cellular defense system. Nrf2 meditates the
expression of genes that encode antioxidant proteins, detoxifying enzymes, metabolic
alteration enzymes and stress response proteins, most of which play critical roles in
regulating cellular defense system, particularly in oxidative stress modulation [26-
28,53]. Through controlling such transcriptional network, Nrf2 is capable of
coordinating a fine-tuned response to multiple stress conditions and detrimental
assaults, maintaining the cellular microenvironmental homeostasis [54]. The Nrf2-
Keap1 complex is a promising therapeutic target for inflammatory disease. Direct
disruption of the Keap1-Nrf2 system could activate the protein levels of Nrf2 and
thereafter display a cell protection molecular mechanism against oxidative attack
when endogenous stress defense mechanisms are imbalanced [55]. Therefore,
improving Nrf2 activity is effective for providing cytoprotection against different
chronic and inflammatory conditions, including PM
2.5
-induced tissue damage
[15,22,24,25]. Nrf2 is involved in the signaling pathway that results in PM-elicited
spermatogenesis dysfunction [56]. In lung cells, Nrf2 protects against diverse PM
components-triggered mitochondrial oxidative damage [57]. Our group also showed
that Nrf2 could alleviate PM
2.5
-induced cardiac injury, hypothalamus dysfunction and
renal damage [15,21,22]. Therapeutic strategy to improve Nrf2 activation has been
suggested to effectively alleviate PM
2.5
-caused pulmonary injury in mice by
suppressing oxidative stress and inflammatory response [58,59]. In our study, we
confirmed that PM
2.5
incubation led to the reduction of Nrf2, suppressing the
expression of anti-oxidant factors, such as HO-1, NQO-1, GCLC, GCLM and SOD,
which are involved in the cellular defense system [60]. After PM
2.5
challenge, Nrf2
-/-
mice exhibited accelerated oxidative damage in liver tissues with lower expression of
anti-oxidants. These data strongly demonstrated that improving Nrf2 activation might
be effective to protect cells and animals from PM
2.5
-induced injury through improving
the anti-oxidative signaling.
SIKE was initially identified as an IKKε-associated protein through yeast two-hybrid
screening, and it is ubiquitously expressed in various tissues [29,30]. The profound
involvement of IKKε in inflammation and immunity demonstrates the potential of
SIKE in regulating the progression of immune diseases [61]. Recently, SIEK was
found to negatively regulate pathological cardiac remodeling through suppressing
protein kinase B (AKT) activation by regulating TBK1. Over-expressing TBK1 was
sufficient to abrogate the protective effects of SIKE on the pathological cardiac
hypertrophy [31]. Furthermore, TBK1 activation has been linked to inflammatory
diseases, such as steatohepatitis [62]. TBK1 plays a modulatory role in the cascade
that results in NF-κB activation and expression of inflammatory factors [32].
Suppressing TBK1 activity could ameliorate fatty liver progression through inhibiting
inflammation [63]. Therefore, improving SIKE to suppress TBK1 might be effective
for inflammation inhibition. In our study, we for the first time demonstrated that
PM
2.5
incubation led to SIKE reduction through a dose- and time-dependent manner
in liver cells, whereas inflammatory signaling TBK1/NF-κB was markedly activated.
The in vivo experiments validated the down-regulation of SIKE in liver of PM
2.5
-
challenged mice. These findings demonstrated the potential of SIKE in controlling
liver damage caused by air pollution. In addition, gain- and loss-function approaches
indicated that SIKE negatively modulated the inflammatory response in PM
2.5
-treated
cells and/or liver tissues. In the absence of SIKE, PM
2.5
-caused expression of IL-1β,
IL-6, TNF-α and IFN-β was further accelerated, along with exacerbated activation of
NF-κB. However, the “rescue” studies in cells showed that SIKE over-expression led
to the striking inhibition of NF-κB activity, repressing inflammation consequently.
Consistent with previous studies, the in vitro studies using the recombinant adenoviral
vector infection analysis demonstrated that SIKE deficiency led to TBK1 over-
activation, subsequently promoting NF-κB activation and inflammation. In contrast,
restoring SIKE in liver cells markedly reduced TBK1 phosphorylation, and then
alleviated inflammation. Importantly, we found that in PM
2.5
-incubated liver cells,
SIKE-restrained inflammatory response and NF-κB activity was largely dependent on
TBK1 activation. These in vitro analysis further demonstrated that SIKE regulated the
development of hepatic injury caused by air pollution mainly through its suppression
to TBK1 signaling.
An important and novel finding here was that Nrf2 exhibited a positive role in
meditating SIKE expression. The in vitro studies using L02 cells showed that Nrf2
knockdown markedly reduced SIKE in liver cells, while promoting Nrf2 rescued
SIKE expression either under normal condition or PM
2.5
stresses. Luciferase reporter
analysis confirmed that Nrf2 positively regulated SIKE expression in HEK-293 cells
stimulated by PM
2.5
. Furthermore, Nrf2 exhibited an inhibitory role in TBK1/NF-κB
activation, restricting inflammatory response eventually, which might be associated
with the improvement of SIKE signal. The in vivo analysis indicated that after long-
term PM
2.5
exposure, SIKE-knockout mice had further decreased expression of anti-
oxidants in liver samples, demonstrating that SIKE might be involved in the
progression of oxidative stress induced by stresses. Therefore, although we illustrated
the regulatory effect of Nrf2 on SIKE, further studies are still require to explore how
Nrf2 regulates SIKE, as well as the potential correlation between the two signaling
pathways.
Juglanin, as natural compound extracted from Polygonum aviculare, exhibits various
biological activities, such as anti-inflammation, anti-cancer and anti-apoptotic cell
death [35,36,38,64]. Given the results that oxidative stress and inflammation played
critical roles in regulating PM
2.5
-induced hepatic injury via Nrf2/SIKE signaling, we
then attempted to find chemoprevention targeting Nrf2 to subsequently ameliorate this
disease. We first showed that Jug boosted Nrf2 protein expression levels and its
nuclear translocation, as well as the down-streaming signals such as HO-1. We then
found that Nrf2 activation by Jug markedly increased the anti-oxidant capacity of L02
cells, as proved by the up-regulated antioxidant enzymes SOD, CAT and GPX. At the
same time, Nrf2 suppressor Keap-1 was decreased in PM
2.5
-stimulated cells. All these
results regulated by Jug decreased cellular ROS in response to PM
2.5
. As expected,
with the improvement of Nrf2, SIKE expression levels were increased in Jug-treated
cells in the absence or presence of PM
2.5
. Consistently, PM
2.5
-caused inflammation
and activation of TBK1/NF-κB were effectively repressed in cells co-treated with Jug.
The in vivo studies using a PM
2.5
-induced chronic hepatic injury model supported the
protective effects of Jug against PM
2.5
-elicited liver damage. Further assessments
showed that Jug significantly attenuated PM
2.5
-induced oxidative stress and
inflammatory conditions by improving Nrf2/SIKE signaling and reducing TBK1/NF-
κB activity.
In summary, as exhibited in Fig. 10, we identified that Nrf2 could improve SIKE
signaling to suppress oxidative stress and inflammatory response induced by PM
2.5
.
Additionally, SIKE-inhibited activation of NF-κB and inflammatory response was
dependent on the blockage of TBK1. Despite more studies are still warranted, the
application of Nrf2/SIKE-induced hepatic protection was verified in mice with long-
term PM
2.5
exposure. Pharmacological intervention analysis further confirmed that
Jug, as a potential activator of Nrf2, suppressed oxidative stress, improved SIKE
expression and then inhibited TBK1 to suppress inflammatory response, alleviating
air pollution-induced chronic hepatic inflammation. Therefore, targeting Nrf2/SIKE
and/or its association with TBK1 may represent promising strategies for developing
effective strategies against PM
2.5
-induced pathological hepatic injury.
Conflict of Interest Statement
The authors see no conflict of interest.
Acknowledgments
This work was supported by (1) National Natural Science Foundation of China
(NSFC Grant No.: 81703527); (2) Chongqing Research Program of Basic Research
and Frontier Technology (Grant No.: cstc2018jcyjA3686, cstc2018jcyjAX0784,
cstc2018jcyjA1472, cstc2018jcyjAX0811, cstc2018jcyjA3533 & KJZD-
M201801601); (3) School-level Research Program of Chongqing University of
Education (Grant No.: KY201710B & 17GZKP01); (4) Advanced Programs of Post-
doctor of Chongqing (Grant No.: 2017LY39); (5) Science and Technology Research
Program of Chongqing Education Commission of China (Grant No.:
KJQN201901608, KJQN201901615 & KJ1601402); (6) Children's Research Institute
of National Center for Schooling Development Programme and Chongqing University
of Education (Grant No.: CSDP19FSO1108) and (7) Chongqing Professional Talents
Plan for Innovation and Entrepreneurship Demonstration Team (CQYC201903258).
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Figure legends
Figure 1. PM
2.5
incubation inhibits Nrf2 and SIKE activation in vitro. (A) RT-
qPCR and western blot results of Nrf2, and (B) RT-qPCR analysis of HO-1, NQO-1,
GCLC and GCLM in L02 cells incubated with 24 h of PM
2.5
(0 to 100 μg/ml as
indicated). (C) RT-qPCR and western blot results of Nrf2, and (D) RT-qPCR results
of HO-1, NQO-1, GCLC and GCLM in L02 cells exposed to PM
2.5
(100 μg/ml) for
the shown time. (E) RT-qPCR and western blot analysis of SIKE, and (F) western blot
results for p-TBK1 and p-NF-κB/p65 in L02 cells treated with different
concentrations of PM
2.5
as indicated for 24 h. (G) RT-qPCR and western blot analysis
of SIKE, and (H) western blot results for p-TBK1 and p-NF-κB in L02 cells cultured
with PM
2.5
(100 μg/ml) for the indicated time. Data are expressed as means ± SEM (n
= 3 independent observations).
*
P < 0.05 and
**
P < 0.01 versus the Con group without
any treatments.
Figure 2. Nrf2 positively regulates SIKE expression in PM
2.5
-incubated cells. (A)
L02 cells were transfected with Nrf2 specific siRNA for 24 h, and then were collected
for transfection efficiency determination using western blotting analysis. (B) RT-
qPCR analysis of HO-1, NQO-1, GCLC and GCLM in L02 cells transfected with
siNrf2 for 24 h. (C) Western blot (left panel) and RT-qPCR (right panel) analysis of
SIKE in L02 cells with siNrf2 transfection for 24 h. (D) Luciferase reporter analysis
with HEK-293 cells that were co-transfected with the indicated reporter plasmids plus
siNC or siNrf2 and then left untreated or treated with PM
2.5
(0, 25, 50 or 100 μg/ml)
for 24 h. (E) L02 cells were transfected with empty plasmid (EP) of Nrf2 plasmids
(Nrf2) for 24 h. Cells were then collected for western blotting analysis to measure
Nrf2 expression levels. (F) RT-qPCR analysis of HO-1, NQO-1, GCLC and GCLM in
L02 cells transfected with Nrf2 plasmids for 24 h. (G) Western blot (left panel) and
RT-qPCR (right panel) analysis of SIKE in L02 cells transfected with 24 h of Nrf2
plasmids. (H) Luciferase reporter analysis with HEK-293 cells transfected with the
indicated reporter plasmids plus EP or Nrf2 plasmids and then left untreated or treated
with increasing concentrations of PM
2.5
for 24 h. (I) L02 cells were treated with PM
2.5
for 24 h, followed by immunofluorescence staining of Nrf2 (green fluorescence) and
SIKE (red fluorescence). Scale bar was 25 μm. (J) Binding of Nrf2 to SIKE by ChIP
assay. L02 cells were treated with or without PM
2.5
(100 μg/ml) for 24 h. Cross-linked
chromatin was immunoprecipitated with an antibody to Nrf2, in the absence of
antibody (input), or an isotype-matched control (IgG). Isolated DNA was purified and
analyzed by PCR. (K) L02 cells were transfected with siNrf2 for 24 h, and were then
incubated with 100 μg/ml of PM
2.5
for another 24 h. Subsequently, all cells were
collected for western blot analysis of SIKE, p-TBK1 and p-NF-κB. (L) L02 cells were
transfected with Nrf2 plasmids for 24 h, followed by PM
2.5
(100 μg/ml) incubation for
another 24 h. Then, western blot analysis was used to determine SIKE, p-TBK1 and
p-NF-κB protein levels. Data are expressed as means ± SEM (n = 3 independent
observations).
*
P < 0.05 and
**
P < 0.01; ns, no significant difference.
Figure 3. Effects of SIKE on TBK1/NF-κB signaling pathway in PM
2.5
-treated
cells. (A) L02 cells were infected with Ad-shRNA, Ad-shSIKE, Ad-GFP or Ad-SIKE
for 24 h. Then, all cells were harvested for western blotting of SIKE expression levels.
L02 cells were infected with Ad-shRNA, Ad-shSIKE, Ad-GFP or Ad-SIKE for 24 h,
and were then subjected to PM
2.5
(100 μg/ml) exposure for another 24 h.
Subsequently, (B) RT-qPCR was used for IL-1β, IL-6, TNF-α and IFN-β mRNA
levels. (C) Western blot analysis was performed for p-TBK1 and p-NF-κB protein
expression levels in whole cells. (D) Western blot analysis of NF-κB in nuclear of
cells as treated. (E) Immunofluorescence staining for NF-κB (green fluorescence) and
p-TBK1 (red fluorescence) in cells. Scale bar was 25 μm. Data are expressed as
means ± SEM (n = 3 independent observations).
*
P < 0.05 and
**
P < 0.01.
Figure 4. SIKE-regulated inflammatory response requires TBK1 blockage in
PM
2.5
-treated cells. L02 cells were infected with Ad-GFP, Ad-SIKE, Ad-TBK1 or
the combination of Ad-SIKE and Ad-TBK1 for 24 h, followed by PM
2.5
(100 μg/ml)
exposure for another 24 h. Then, (A) RT-qPCR was used to determine IL-1β, IL-6,
TNF-α and IFN-β mRNA levels. (B) Western blot analysis was conducted for cellular
p-NF-κB. L02 cells were infected with Ad-shRNA, Ad-shSIKE, Ad-shTBK1 or the
combination of Ad-shSIKE and Ad-shTBK1 for 24 h, followed by PM
2.5
(100 μg/ml)
incubation for another 24 h. Next, all cells were harvested for further experiments. (C)
RT-qPCR analysis of IL-1β, IL-6, TNF-α and IFN-β in cells. (D) Western blot
analysis for p-NF-κB protein expression levels. Data are expressed as means ± SEM
(n = 3 independent observations).
*
P < 0.05 and
**
P < 0.01.
Figure 5. Juglanin improves Nrf2 activation to inhibit oxidative stress in PM
2.5
-
incubated cells. (A) L02 cells were treated with different concentrations of Jug (0 to
80 μM) for 24 h. Then, morphology of cells was observed. (B) Left, L02 cells were
incubated with Jug at the indicated concentrations for 24 h, and were then collected
for cell viability calculation using western blotting analysis. Right, L02 cells were
treated with 40 μM of Jug for the shown time, followed by MTT analysis to assess the
cell viability. (C) Western blotting results for nuclear and cytoplasm Nrf2 expression
levels in L02 cells incubated with increasing concentrations of Jug for 24 h. (D)
Immunofluorescence staining of Nrf2 in Jug-treated L02 cells for 24 h. Scale bar was
25 μm. (E-G) L02 cells were treated with PM
2.5
(100 μg/ml) for 24 h together with
Jug (40 μM) or t-BHQ (10 μM). Then, all cells were collected for further analysis as
follows. (E) Western blot analysis of Keap-1 and HO-1. (F) Assessments of SOD,
CAT and GPX activities in cells. (G) DCF-DA staining of L02 cells. Data are
expressed as means ± SEM.
*
P < 0.05 and
**
P < 0.01; ns, no significant difference.
Figure 6. Juglanin treatment suppresses inflammation in PM
2.5
-incubated cells
through increasing SIKE expression. L02 cells were treated with PM
2.5
(100 μg/ml)
for 24 h together with Jug (40 μM) or t-BHQ (10 μM). Then, all cells were harvested
for the following studies. (A) Western blot analysis of SIKE in cells. (B)
Immunofluorescence staining of SIKE in cells. Scale bar was 25 μm. (C) RT-qPCR
analysis of IL-1β, IL-6, TNF-α and IFN-β in cells. (D) Western blot analysis for p-
TBK1 and p-NF-κB protein expression levels. Data are expressed as means ± SEM (n
= 3 independent observations).
*
P < 0.05,
**
P < 0.01 and
***
P < 0.001; ns, no
significant difference.
Figure 7. Effects of SIKE expression on PM
2.5
-induced hepatic injury in mice. (A)
Body weight change of mice (n = 8 per group). (B) MBP results were shown (n = 8
per group). (C) H&E staining of liver sections (n = 5 per group). Scale bar was 50 μm.
(D) Liver weight (n = 8 per group). (E) ELISA results for IL-1β, IL-6 and TNF-α in
liver samples (n = 8 per group). (F) RT-qPCR of hepatic IL-1β, IL-6, TNF-α and IFN-
β mRNA expression levels (n = 5 per group). (G) Western blot analysis of p-TBK1
and p-NF-κB in liver samples (n = 5 per group). (H) RT-qPCR analysis of HO-1,
NQO-1, GCLC and GCLM in liver samples (n = 5 per group). (I) Hepatic SOD
activity and MDA levels (n = 8 per group). Data are expressed as means ± SEM.
*
P <
0.05 and
**
P < 0.01.
Figure 8. Effects of Nrf2 activation on PM
2.5
-induced hepatic injury in mice. (A)
Body weight of mice was recorded (n = 8 per group). (B) MBP results for mice from
each group (n = 8 per group). (C) Liver weight of mice (n = 8 per group). (D) H&E
staining of hepatic sections (n = 5 per group). Scale bar was 50 μm. (E) SOD and
MDA measurements in liver samples (n = 8 per group). (F) RT-qPCR analysis of HO-
1, NQO-1, GCLC and GCLM in liver tissues. (G) Immunofluorescence staining of
SIKE in hepatic sections (n = 5 per group). Scale bar was 50 μm. (H) Quantification
of SIKE fluorescence intensity. (I) Western blot results for SIKE in liver samples (n =
5 per group). (J) RT-qPCR results for hepatic IL-1β, IL-6, TNF-α and IFN-β mRNA
expression levels (n = 5 per group). (K) Western blot analysis of liver p-TBK1 and p-
NF-κB (n = 5 per group). Data are expressed as means ± SEM.
*
P < 0.05 and
**
P <
0.01; ns, no significant difference.
Figure 9. Juglanin ameliorates PM
2.5
-induced hepatic injury through improving
Nrf2/SIKE signaling pathway in mice. (A) Body weight change of mice (n = 8 per
group). (B) MBP results for mice (n = 8 per group). (C) Liver weight of mice (n = 8
per group). (D) Up panel, H&E staining for liver samples; down panel,
immunofluorescence staining of Nrf2 (green) and SIKE (red) in liver sections (n = 5
per group). Scale bar was 50 μm. (E) Western blot analysis for liver Nrf2, SIKE, p-
TBK1 and p-NF-κB (n = 5 per group). (F) RT-qPCR results for hepatic Nrf2 mRNA
expression levels (n = 5 per group). (G) SOD and MDA in liver tissues were
measured (n = 8 per group). (H) RT-qPCR results for hepatic IL-1β, IL-6, TNF-α,
IFN-β, HO-1, NQO-1, GCLC and GCLM (n = 5 per group). Data are expressed as
means ± SEM.
*
P < 0.05 and
**
P < 0.01; ns, no significant difference.
Figure 10. Proposed mechanism of Nrf2/SIKE-mediated hepatic injury induced
by long-term PM
2.5
exposure. After PM
2.5
long-term challenge, down-regulated
activation of Nrf2 repressed SIKE expression, contributing to oxidative stress and
inflammatory response through reducing anti-oxidants and facilitating TBK1/NF-κB
signaling. These effects led to hepatic injury consequently. However, Juglanin
treatment showed protective effects against PM
2.5
-induced liver injury by improving
Nrf2/SIKE signaling pathway.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Highlights
1. PM
2.5
incubation inhibits Nrf2 and SIKE activation in vitro.
2. Nrf2 positively regulates SIKE expression in PM
2.5
-incubated cells.
3. SIKE-regulated inflammatory response requires TBK1 blockage in PM
2.5
-treated
cells.
4. Juglanin treatment suppresses inflammation in PM
2.5
-incubated cells through
increasing SIKE expression.
Conflict of Interest
The authors see no any conflict of interest.
